The ultimate goal of this proposal is to obtain mice heterozygous for mutations in specific genes thought to play an important role in the control of normal embryonic development. We propose two alternative plans for obtaining such mice. Both involve the creation of insertion mutations in the particular gene of interest in mouse embryonic stem (ES) cells in vitro. One plan employs a method that is termed "gene replacement," whereby we will attempt to target the insertion into the gene of interest by injecting into ES cells a mutated segment of that gene, and then isolate cells in which the incoming, mutated DNA has replaced one of the endogenous copies of that gene segment. The other plan involves the establishment of a library of frozen male ES cells in which "saturation mutagenesis" has occurred following retroviral integration. This library will be screened by the "polymerase chain reaction" (PCR) method to identify and isolate individual ES cell lines heterozygous for a proviral insertion in the gene of interest, once cells with mutations in the gene of interest are obtained, they will be injected into mouse blastocysts, which will be placed in foster mothers and allowed to develop to term. Any chimeric mice that are born will be mated to determine if the mutated ES cells have colonized the germ line. The successful conversion of mutated ES cells into functional germ cells would allow the establishment of new strains of mice heterozygous for an insertion mutation in the gene of interest. Once such mice are available, we would use them to obtain, by breeding, mutant homozygous embryos, which could then be analyzed to determine the effects on development of a lack of the wild-type gene product. The information gained from such studies could lead, ultimately, to an understanding of the functions of these genes in development.